October 21-25, 2013: Inhibition Assay I October 26, 2013: Rice Undergraduate Research Symposium November 2, 2013: Virtual screening against novel compounds: ChemBridge-diversity3D library
Figure 1. NDM-1 inhibition assay I results. 'In house' compound 5107893 tested in 5 minute time-based assay. Nitrocefin added after approx. 30 seconds. Normal nitrocefin hydrolase activity at 486 nm indicates no inhibition of NDM-1. Additional concentration to be tested.
'In house' top-ranking compounds were acquired. Compound 5107893 was tested for inhibition in an enzyme assay. Inhibitory activity was not indicated, as normal nitrocefin hydrolase activity was detected at 486 nm. Additional concentration of compound 5107893 will be tested, along with other top-ranking 'in house' compounds. Top ranking cb306 compounds will be ordered. Virtual screening round against ChemBridgediversity3D library is currently being completed.
Weeks 7 & 8: October 7 - October 20 Have you collected any more data for weeks 7 & 8? Thank you. -Max 10/21/13
October 10, 2013: Synergy HT Biotek Reader to determine optimal [NDM-1] October 11, 2013: Determining control set of ligands October 14-18, 2013:LigPrep protocol, GOLD & ICM control ligands docking, virtual screening against novel compounds: cb306 library & 'in house' library
Table 4. Virtual screening against novel compounds: top 7 'in house' library ligands. Best GOLD fitness scores 80-89.55. Compounds ranked 1, 3, 4, and 7 pursued further with favorable LogP values (<2.7).
Table 3. Virtual screening against novel compounds: top 14 cb306 library ligands. Four compounds with GOLD fitness scores >90. All top 14 ligands follow Lipinski's rule of 5. Compounds ranked 1, 2, 5, 9, and 11 pursued further with favorable LogP values (<2.4).
Table 2. ICM validation control docking results. 11 positive control ligands and 5 negative control ligands (ZINC) docked against NDM-1 PDB ID: 4EY2 (chain A). Positive controls are highlighted in green and negative controls are highlighted in pink. Positive and negative control ligands are interspersed, with a few unexpected results. GOLD evaluated control ligands more accurately.
Table 1. GOLD validation control docking results. 11 positive control ligands and 5 negative control ligands (ZINC) docked against NDM-1 PDB ID: 4EY2 (chain A). Positive controls are highlighted in green and negative controls are highlighted in pink. All positive control ligands scored higher than negative control ligands as expected, thus validating GOLD virtual screening software.
Positive control ligands were acquired from PubChem after referring to literature. Negative control ligands were established by the Zinc database after inputting physico-chemical properties of positive control ligands. These ligands were ligprepped and control ligand docking on GOLD & ICM was conducted. NDM-1 PDB ID: 4EY2 was utilized and prepared in GOLD Hermes (2254 Hs added, 227 H2Os deleted). GOLD scored control ligands more accurately than ICM. Virtual screening against novel compounds was conducted using cb306 and 'in house' libraries. Potential inhibitors from both libraries will be acquired and tested for inhibition concurrently.
Weeks 5 & 6: September 23 - October 6
Great Captions and Great analysis. Keep up the nice work. Thank you. -Max 10/07/2013 Good Results Serena, keep up the good work! - Dr. B
September 26-27, 2013: Optimal substrate (nitrocefin) dilution to establish absorbance signal, NDM-1 enzyme assay II September 30, 2013: NDM-1 enzyme assay III :) October 1, 2013: NDM-1 1.5 hour enzyme assay IV October 3-4, 2013: Virtual screening: ICM control docking
Figure 4. ~100 minute time-based NDM-1 enzyme assay IV (conducted on 10/1/2013 using spec: Luke) using 2 ug un-diluted NDM-1 (aliquot 1), 1:5 nitrocefin, buffer B, and autoclaved nanopure water. Substrate added after 1 minute. Absorbance monitored at both 390.0 and 486.0 nm. Rapidly increasing, linear absorbance signal at 486.0 nm indicates hydrolase activity of functional NDM-1 enzyme. 390.0 nm absorbance expected to decrease but fluctuated near 3.0 absorbance throughout assay.
Figure 3. ~30 minute time-based NDM-1 enzyme assay III (conducted on 9/30/2013 using spec: Chipper) using 2 ug un-diluted NDM-1 (aliquot 1), 1:5 nitrocefin, buffer B, and autoclaved nanopure water. Substrate added after 1 minute. Absorbance monitored at both 390.0 and 486.0 nm. Rapidly increasing absorbance signal at 486.0 nm indicates hydrolase activity of functional NDM-1 enzyme! 390.0 nm absorbance expected to decrease but fluctuated above 1.5 absorbance throughout assay.
Figure 2. 10 minute time-based enzyme assay II LoggerPro results using 1:1000 NDM-1 enzyme, 1:5 nitrocefin dilution, buffer B, and autoclaved nanopure water. Absorbance change is insignificant. Greater concentration of NDM-1 needed.
Figure 1. Full spectrum of varied nitrocefin substrate concentrations. Low absorbance at 485 nm indicates nitrocefin is functional. Optimal absorbance signal for time-based enzyme assay is 0.5-1.0 at 485 nm, achieved using 1:5 nitrocefin dilution.
Optimal substrate (nitrocefin) dilution was established to register absorbance signal during enzyme assays. 1:5 nitrocefin dilution determined using results from Fig. 1 above (optimal absorbance of 0.5-1.0 at 485 nm). 1:5 substrate dilution was utilized during the second attempt of the enzyme assay. Results in Fig. 2 illustrate that the absorbance change was insignificant during the 10 minute time-based assay, as NDM-1 dilutions created assay conditions with too little enzyme. Enzyme assay III was successful after using 2ug un-diluted NDM-1, which resulted in an expected increase in absorbance through NDM-1 hydrolase activity. NDM-1 enzyme assay results (9/30-10/1/2013) validate reproducibility of assay data for same aliquot of NDM-1. Future research will involve determining optimal [NDM-1] to create a plot with varied substrate concentrations to ultimately determine the Km value. In addition, other aliquots of NDM-1 will be tested to ensure functionality. Lastly, these results indicate that the NDM-1 enzyme purified on 7/31/2013 is indeed functional and can be utilized for enzyme inhibition assays after virtual screening is completed.
Week 3 & 4: September 9 - September 22 Serena - ok good. Show a pretty picture (screenshot is ok) of an enzyme assay graph though. Dr. B 100113
September 12, 2013: Planning NDM-1 enzyme assay September 13, 2013:Prepared buffers: NDM-1 enzyme assay September 19, 2013: NDM-1 enzyme assay I with substrate nitrocefin
Preparation of assay buffers
Source: Thomas, P.; Zheng, M.; Wu, S.; Guo, H.; Liu, D.; Xu, D.; Fast, W., Characterization of Purified New Delhi Metallo-β-lactamase-1. Biochemistry 2011, 50, (46), 10102-10113.
Dilute NDM-1 in Buffer A: 50 mM Tris-HCl, pH 7 and 150 mM NaCl Buffer B: 50 mL 0.1 M Hepes, pH 7 and 20 µM ZnSO4 solution 0.1 mM colorimetric nitrocefin substrate in water (VWR, Radnor, Pennsylvania) from 100 mM stock solution of 5 mg nitrocefin in DMSO Prepare 0.16-0.33 µg of NDM-1 in total volume of 300 µL and monitor product absorbance at ~485nm
Results of NDM-1 enzyme assay I with nitrocefin:
Spectrophotometric time-based assay (spec: Chipper) resulted in 0 absorbance signal at 486.00 nm for samples: Buffer B only, Buffer B + nitrocefin, and 4 µL NDM-1 enzyme + buffer B + nitrocefin.
Full spectrum assay conducted and illustrated 0 absorbance for buffer B only. 0.02 absorbance at ~400 nm and 0 absorbance for all other wavelengths observed for buffer B + nitrocefin sample. In addition, the following samples were tested at full spectrum and had an insignificant absorbance output (maximum 0.01 absorbance at 400 nm): 4 µL NDM-1 + buffer B + nitrocefin, 5 µL NDM-1 + buffer B + nitrocefin, 20 µL NDM-1 + buffer B + nitrocefin, 80 µL NDM-1 + buffer B + nitrocefin, 90 µL NDM-1 + buffer B + nitrocefin, and Cuvette with Vf = 600 µL of NDM-1 + buffer B + nitrocefin.
These results are worrisome and do not correlate with the cell-based overnight NDM-1 growth assay results. Spectrophotometric signal should result with nitrocefin. Substrate amount may be too minimal to register an absorbance signal. In addition, these results indicate that the NDM-1 enzyme may be inactive (purified 7/31/13). The ExPASy ProtParam tool establishes the estimated half-life of NDM-1: 30 hours (mammalian reticulocytes, in vitro), >20 hours (yeast, in vivo), and >10 hours (Escherichia coli, in vivo). A second enzyme assay should be conducted with increased substrate concentration along with a second spec to elucidate accuracy of Chipper. An additional cell-based overnight NDM-1 growth assay should be completed to test whether the NDM-1 protein is still active. If results are negative, a new batch of purified NDM-1 will need to be utilized.
Serena, ok good. Dr. B 090913
Week 1 & 2: August 26 - September 8 August 29, 2013: Cell-based overnight NDM-1 growth assay August 30, 2013: Results of overnight NDM-1 growth assay
Results:
2 transformation tubes (+Kan, -Amp): bacterial growth 2 transformation tubes (+ Kan, +Amp): no bacterial growth 2 transformation tubes (+Kan, +Amp, 250 uL NDM-1 protein): medium bacterial growth 2 transformation tubes (+Kan, +Amp, 500 uL NDM-1 protein): large bacterial growth 2 transformation tubes (+Kan, +Amp, 1.5 mL NDM-1 protein): no bacterial growth
Transformation tubes post 16 hours in shaking incubator displayed, for the most part, expected results. However, 1.5 mL NDM-1 protein tubes should have theoretically contained greater bacterial growth than tubes containing 250 uL and 500 uL NDM-1. These results provide confidence that the NDM-1 protein is functional.
July 29, 2013: ----- July 30, 2013: NDM-1 protein expression (day V) - sonication & spin down July 31, 2013: NDM-1 Ni-NTA affinity purification August 1, 2013: ----- August 2, 2013: NDM-1 protein characterization; gel dried on 8/5/2013 - K.R.
Figure 2. NDM-1 SDS-PAGE gel post-staining with recycled imperial protein stain for 90 minutes on orbital shaker. Lane 1 & 2 = empty, lane 3 = PAGE gel ruler, lane 4 = sample 2 (soluble fraction), lane 5 = sample 3 (flow through), lane 6 = sample 4 (wash), lane 7 = sample 5 (elution 1), lane 8 = sample 6 (elution 2), lane 9 & 10 = empty. Presence of one thick band in elution 1 lane at ≈ 30 kDa (between 25-35 kDa marks - refer to Figure 1 below) indicates with relative certainty a high yield and purity of NDM-1 protein (MW 28.6 kDa). Dried SDS-PAGE gel illustrates very faint presence of other bands in elution 1 lane.
Figure 1. SDS-PAGE band profile of the Thermo Scientific PageRuler Prestained Protein Ladder for 4-20% Tris-glycine gel
July 22, 2013:Results available - cloning #2; starter culture of transformed BL21(DE3) E. coli with pET27b(+) NDM-1(d35): protein expression (day II) July 23, 2013: NDM-1 protein expression (day III) - K.R. July 24, 2013: ----- July 25, 2013: Preparation of purification lysis, wash, & elution buffers July 26, 2013: -----
Table 2. pNIC-Bsa4 cloning round 2 with NDM-1 insert results. Sample #, primer, query coverage (%), identities (#, %), pos/neg clone, and notes provided for each sample submitted to DNA sequencing facility. Information obtained from aligning 2 sequences on NCBI's nucleotide BLAST: core's DNA sequence results (query) and NDM-1 gene DNA sequence (subject). + clone from sample #8!
+ Clone Pairwise Alignments & Chromatograms
Figure 4. Forward read nucleotide BLAST pairwise alignment between core's DNA sequence results for sample #8 (query) and known NDM-1 gene DNA sequence (subject). 65% query coverage and 812/813 (99%) identities. Contains complete coding DNA sequence (813 nt). 1 N at bp 896 (insignificant) - analyzed in chromatogram below. Sample #8 forward read is a positive clone.
Figure 3. Chromatogram for sample #8 forward read provided by core. Query contains N instead of A, C, T, or G at bp 896 in Figure 4. Chromatogram illustrates slightly dominant blue peak aligning with N at position 896. Cytosine signal present - N most likely due to simultaneous guanine signal from bps 894-895. C matches NDM-1 gene sequence, shown above in Figure 4. Sample #8 forward read is a positive clone as every base pair matches accurately with known NDM-1 sequence. Zero deletions, insertions, point mutations present. Sample #8 can be used for protein expression.
Figure 2. Reverse read nucleotide BLAST pairwise alignment between core's DNA sequence results for sample #8 (query) and known NDM-1 gene DNA sequence (subject). 64% query coverage and 812/813 (99%) identities. Contains complete coding DNA sequence (813 nt). 1 N at bp 868 (insignificant) - analyzed in chromatogram below. Sample #8 reverse read is a positive clone
Figure 1. Chromatogram for sample #8 reverse read provided by core. Query contains N instead of A, C, T, or G at bp 868 in Figure 2. Chromatogram illustrates dominant black peak aligning with N at position 868. Guanine signal present - N most likely due to simultaneous, weak adenine signal from bp 867. G matches NDM-1 gene sequence, shown above in Figure 2. Sample #8 reverse read is a positive clone as every base pair matches accurately with known NDM-1 sequence. Zero deletions, insertions, point mutations present. Sample #8 can be used for protein expression.
pNIC-Bsa4 Cloning #2 with NDM-1 Insert Nanodrops
Colony #1. 2uL pNIC-Bsa4 cloned with NDM-1 insert eluted in 50 uL Elution Buffer. 260/280 = 1.99, 260/230 = 1.70, Concentration = 87.1 ng/uL. Purity ratios are within 0.3 range of 'pure' values. Great concentration - sample submitted to DNA sequencing facility.
Colony #2. 2uL pNIC-Bsa4 cloned with NDM-1 insert eluted in 50 uL Elution Buffer. 260/280 = 1.94, 260/230 = 1.65, Concentration = 107.8 ng/uL. 260/230 ratio could be improved. Excellent concentration - sample submitted to DNA sequencing facility.
Colony #3. 2uL pNIC-Bsa4 cloned with NDM-1 insert eluted in 50 uL Elution Buffer. 260/280 = 2.02, 260/230 = 1.79, Concentration = 79.3 ng/uL. Purity ratios are within 0.2 range of 'pure' values. Great concentration - sample submitted to DNA sequencing facility.
Colony #4. 2uL pNIC-Bsa4 cloned with NDM-1 insert eluted in 50 uL Elution Buffer. 260/280 = 2.04, 260/230 = 1.99, Concentration = 76.0 ng/uL. Purity ratios are within 0.25 range of 'pure' values. Good concentration - sample submitted to DNA sequencing facility.
Colony #5. 2uL pNIC-Bsa4 cloned with NDM-1 insert eluted in 50 uL Elution Buffer. 260/280 = 1.98, 260/230 = 1.72, Concentration = 82.7 ng/uL. Purity ratios are within 0.3 range of 'pure' values. Great concentration - sample submitted to DNA sequencing facility.
Colony #6. 2uL pNIC-Bsa4 cloned with NDM-1 insert eluted in 50 uL Elution Buffer. 260/280 = 2.02, 260/230 = 1.89, Concentration = 69.5 ng/uL. Purity ratios are within 0.2 range of 'pure' values. Good concentration - sample submitted to DNA sequencing facility.
Colony #7. 2uL pNIC-Bsa4 cloned with NDM-1 insert eluted in 50 uL Elution Buffer. 260/280 = 2.00, 260/230 = 1.84, Concentration = 64.4 ng/uL. Purity ratios are within 0.2 range of 'pure' values. Good concentration - sample submitted to DNA sequencing facility.
Colony #8. 2uL pNIC-Bsa4 cloned with NDM-1 insert eluted in 50 uL Elution Buffer. 260/280 = 1.99, 260/230 = 1.53, Concentration = 72.5 ng/uL. 260/230 purity could be improved. Good concentration - sample submitted to DNA sequencing facility.
Colony #9. 2uL pNIC-Bsa4 cloned with NDM-1 insert eluted in 50 uL Elution Buffer. 260/280 = 2.00, 260/230 = 1.92, Concentration = 65.5 ng/uL. Purity ratios are within 0.2 range of 'pure' values. Good concentration - sample submitted to DNA sequencing facility.
NOTE: Samples 1-9 eluted correctly in 50 uL Elution Buffer. All Nanodrops illustrate lower concentration value of 2 trials completed for each colony.
July 14, 2013: Spin down 8 cloning samples July 15, 2013: Mini-Prep 8 cloning samples July 16, 2013: Submit 10 cloning samples - DNA sequencing facility July 17, 2013: Results available - cloning #1 July 18, 2013:Prepared overnight culture of 8 colonies from Masterplate A - cloning #2 July 19, 2013: Submit Midi-PreppedpET27b(+) NDM-1(d35) samples - DNA sequencing facility; spin down, Mini-Prep, submit 9 cloning #2 samples
Table 1. pNIC-Bsa4 cloning round 1 with NDM-1 insert results. Sample #, primer, query coverage (%), identities (#, %), pos/neg clone, and notes provided for each sample submitted to DNA sequencing facility. Information obtained from aligning 2 sequences on NCBI's nucleotide BLAST: core's DNA sequence results (query) and NDM-1 gene DNA sequence (subject). No + clones - perform round 2 of cloning.
NOTE: Changes for round 2 of cloning: elute in accurate amount of Elution Buffer (50 uL); use correct transformation tubes; when eluting - insert buffer directly into the center of column to maximize amount of eluted DNA.
pNIC-Bsa4 Cloning #1 with NDM-1 Insert Nanodrops
Colony #1.2uL pNIC-Bsa4 cloned with NDM-1 insert eluted in 100 uL Elution Buffer. 260/280 = 1.70, 260/230 = 0.93, Concentration = 70.6 ng/uL. 260/230 purity could be greatly improved. Good concentration - sample submitted to DNA sequencing facility.
Colony #2.2uL pNIC-Bsa4 cloned with NDM-1 insert eluted in 100 uL Elution Buffer. 260/280 = 1.92, 260/230 = 1.29, Concentration = 41.3 ng/uL. 260/230 purity could be improved. OK concentration - sample submitted to DNA sequencing facility.
Colony #3.2uL pNIC-Bsa4 cloned with NDM-1 insert eluted in 100 uL Elution Buffer. 260/280 = 2.07, 260/230 = 1.66, Concentration = 35.6 ng/uL. 260/230 and 260/280 ratios could be improved. OK concentration - sample submitted to DNA sequencing facility.
Colony #4.2uL pNIC-Bsa4 cloned with NDM-1 insert eluted in 100 uL Elution Buffer. 260/280 = 2.04, 260/230 = 1.70, Concentration = 33.2 ng/uL. 260/230 and 260/280 ratios could be improved. OK concentration - sample submitted to DNA sequencing facility.
Colony #5.2uL pNIC-Bsa4 cloned with NDM-1 insert eluted in 100 uL Elution Buffer. 260/280 = 1.93, 260/230 = 1.52, Concentration = 40.2 ng/uL. 260/230 purity could be improved. OK concentration - sample submitted to DNA sequencing facility.
Colony #6.2uL pNIC-Bsa4 cloned with NDM-1 insert eluted in 100 uL Elution Buffer. 260/280 = 1.96, 260/230 = 1.81, Concentration = 18.3 ng/uL. 260/230 purity could be improved. Very low concentration - sample submitted to DNA sequencing facility.
Colony #7.2uL pNIC-Bsa4 cloned with NDM-1 insert eluted in 100 uL Elution Buffer. 260/280 = 1.79, 260/230 = 1.11, Concentration = 45.5 ng/uL. 260/230 purity could be improved. OK concentration - sample submitted to DNA sequencing facility.
Colony #8. 2uL pNIC-Bsa4 cloned with NDM-1 insert eluted in 100 uL Elution Buffer. 260/280 = 1.75, 260/230 = 0.95, Concentration = 56.3 ng/uL. 260/230 purity could be improved. Good concentration - sample submitted to DNA sequencing facility.
NOTE: Samples 1-8 eluted mistakenly in 100 uL Elution Buffer - incorrectly referred to Mini-Prep kit manual. Should elute in 50 uL Elution Buffer. This most likely caused low, non-ideal concentration values in above Nanodrops, particularly for Colony #6.
July 8, 2013: Secondary PCR, 1° and 2° PCR agarose gel electrophoresis verification, pNIC-Bsa4 overnight culture preparation, Naturally Obsessed: The Making of a Scientist documentary viewing July 9, 2013: PCR squared reaction, PCR cleanup, pNIC-Bsa4 Midi-Prep - submitted to DNA sequencing facility July 10, 2013: Cloning: preparation of pNIC-Bsa4 as accepting vector + PCR cleanup, YopH spin down, transformation of DH5α & BL21(DE3) E.coli with pET27b(+) NDM-1(d35) (Day I) July 11, 2013: pNIC-Bsa4 preparation I gel check, preparation of pNIC-Bsa4 II & III, PCR combination cleanup, gel check II, FPLC - ECDHFR, overnight culture of DH5α E.coli with pET27b(+) NDM-1(d35) (Day II) July 12, 2013:Spin down & Midi-Prep ofDH5α E.coli with pET27b(+) NDM-1(d35) (Day III), cloning: cohesive end generation on PCR insert & accepting vector, annealing & transformation step July 13, 2013: Master plate & overnight culture of 8 colonies
DNA Sequencing Results: pET27b(+) NDM-1(d35) forward sequence post-Midi-Prep
Figure 8. NCBI Nucleotide BLAST (Basic Local Alignment Search Tool) significant alignments of DNA sequencing results for midi-prepped pET27b(+) NDM-1(d35). 58% query coverage and 100% identities with Klebsiella pneumoniae strain NF23142 metallo-beta-lactamase NDM-1 (blaNDM-1) gene, complete cds.
Figure 7. NCBI Nucleotide BLAST pairwise alignment between significant alignment of DNA sequencing results for midi-prepped pET27b(+) NDM-1(d35) and Klebsiella pneumoniae strain NF23142 metallo-beta-lactamase NDM-1 (blaNDM-1) gene, complete cds. Identities = 705/705 (100%).
pET27b(+) NDM-1(d35) Transformed in DH5α E. coli Midi-Prep Nanodrops
Trial 2.260/280 = 1.94, 260/230 = 3.37, Concentration = 41.5 ng/uL. Purity could be improved. Sample submitted to DNA sequencing facility.
Trial 1.260/280 = 1.98, 260/230 = 3.15, Concentration = 42.4 ng/uL. Purity could be improved. Sample submitted to DNA sequencing facility.
Figure 6. Agarose gel electrophoresis image of pNIC-Bsa4 preparation II/III as accepting vector restriction enzyme digest with BsaI-HF. Lane 1 = skip, lane 2 = molecular weight marker 1kb DNA ladder, lane 3 = 15 uL sample of cut pNIC-Bsa4 post PCR cleanup, lanes 4-6 = skip. Bands appear identical to expected results from custom digest virtual gel (right image) created by NEB cutter – 1 distinct band at 2kb and 1 at 5kb-6kb – confirming pNIC-Bsa4 is prepared as accepting vector.
Preparation #2, 3 of pNIC-Bsa4 as Accepting Vector post-PCR combination cleanup Nanodrops Trial 2.260/280 = 1.90, 260/230 = 2.31, Concentration = 65.6 ng/uL. Sample of pNIC-Bsa4 considered 'pure'; includes prep #1 (20 uL), prep #2 (50 uL), prep #3 (50 uL) for a Vf = 120 uL pNIC-Bsa4. Combination sample is usable as an accepting vector.
Trial 1. 260/280 = 1.87, 260/230 = 2.14, Concentration = 63.3 ng/uL. Sample of pNIC-Bsa4 considered 'pure'; includes prep #1 (20 uL), prep #2 (50 uL), prep #3 (50 uL) for a Vf = 120 uL pNIC-Bsa4. Combination sample is usable as an accepting vector.
Figure 5. Agarose gel electrophoresis image of pNIC-Bsa4 preparation as accepting vector restriction enzyme digest with BsaI-HF. Lane 1 = skip, lane 2 = molecular weight marker 1kb DNA ladder, lane 3 = 15 uL sample of cut pNIC-Bsa4 post PCR cleanup, lanes 4-6 = skip. Bands appear identical to expected results from custom digest virtual gel (right image) created by NEB cutter – 1 distinct band at 2kb and 1 at 5kb-6kb – confirming pNIC-Bsa4 is prepared as accepting vector.
Figure 4. 1 kb DNA Ladder visualized by ethidium bromide staining on a 0.8% TAE agarose gel
Preparation of pNIC-Bsa4 as Accepting Vector post-PCR cleanup Nanodrops
Figure 3. NCBI Nucleotide BLAST (Basic Local Alignment Search Tool) significant alignments of DNA sequencing results for midi-prepped pNIC-Bsa4. 82% query coverage and 99% identities with expression vector pNIC28-Bsa4, complete sequence. Prepared pNIC-Bsa4 will serve as a suitable protein expression vector, and can be prepped as accepting vector.
pNIC-Bsa4 PCR cleanup Nanodrops
Elution 1 Trial 2
Elution 1 Trial 1
PCR^2 cleanup Nanodrops
Elution 1 Trial 2
Elution 1 Trial 1
Figure 2. Agarose gel electrophoresis image 1° and 2° overlap PCR of K. pneumoniae New Delhi metallo-β-lactamase 1 gene using custom designed forward and reverse primers (ordered 7/1/2013). Lane 1 = skip, lane 2 = molecular weight marker 100bp DNA ladder, lane 3 = 15 uL 1° PCR, lane 4 = 15 uL 2° PCR, lane 5 & 6 = skip. PCR and oligo mix confirmed by 1° PCR smear and strong, distinct 2° PCR band corresponding to size of NDM-1 CDS (813 nt).
Figure 1. 100 bp DNA Ladder visualized by EtBr staining on a 1.3% TAE agarose gel
July 1, 2013: Protocol primer design - pNIC-BSA4 - New Delhi metallo-beta-lactamase-1 (computer lab) July 2, 2013: Protein characterization - drying the SDS-PAGE gel, Restriction enzyme digest - preparing samples July 3, 2013: Restriction enzyme digest - agarose gel electrophoresis verification, PCR protocol for pLIC sequencing vectors of pNIC-Bsa4 July 4, 2013: Holiday July 5, 2013: pLIC PCR - agarose gel electrophoresis verification, Primer overlap PCR with Q5 Polymerase - primer dilutions for assembly step July 6, 2013: Optional session - Primary PCR
Figure 4. Agarose gel electrophoresis image pNIC-Bsa4 plasmid post-PCR using pLIC forward and reverse primers. Lane 1 = skip, lane 2 = molecular weight marker 100 bp DNA ladder, lane 3 = sample 1 (1:1000 dilution 3, 0.035 ng/uL pNIC-Bsa4), lane 4 = sample 2 (1:100 dilution 2, 0.35 ng/uL pNIC-Bsa4), lane 5 = sample 3 (1:10 dilution 1, 3.5 ng/uL pNIC-Bsa4), lane 6 = control, sample 4 (0 ng/uL pNIC-Bsa4), lanes 6-10 = empty. Presence of bands in lanes 3-5 illustrate proper amplification of DNA, but band in control lane 5 indicates contamination. Increasing intensity of bands from lanes 3-5 demonstrate higher concentrations of plasmid pNIC-Bsa4 DNA.
Figure 3. Lanes 1-5: K.K. overlap primer PCR, lanes 6-12: S.Z. restriction enzyme digest of pGBR22 plasmid. Lane 6 = skip, lane 7 = molecular weight marker 1kb DNA ladder, lane 8 = uncut pGBR22 plasmid, lane 9 = EcoRI digestion reaction, lane 10 = PvuII digestion reaction, lane 11 = EcoRI + PvuII digestion reaction, lane 12 = skip. Bands appear identical to expected results from virtual gel (right image) created by NEB cutter in the “Analyze DNA Sequence” protocol from 6/24/2013.
Figure 2. Dried SDS-PAGE gel post-protein characterization of YopH. Lane 1 = empty, lane 2 = PAGE gel ruler, lane 3 = sample 0 (cell lysate before induction), lane 4 = sample 1 (cell lysate after induction), lane 5 = sample 2 (soluble fraction), lane 6 = sample 3 (flow through), lane 7 = sample 4 (wash), lane 8 = N/A (incorrectly added samples 4 & 5 – skip), lane 9 = sample 5 (elution 1), lane 10 = sample 6 (elution 2). Presence of thick band across gel (boxed) and specifically from elution 1 lane at ≈ 30 kDa (between 25-35 kDa marks) indicates with relative certainty a high yield and purity of YopH (MW of 33,512.8)
Figure 1. New Delhi metallo-beta-lactamase-1 primer design - pNIC-BSA4 with 5'---3' forward and reverse primers and melting temperatures
June 24, 2013: Analyze DNA sequence exercise (computer lab) June 25, 2013: Protein expression scaled up - day 5: sonication & spin down steps June 26, 2013: Protein purification, SDS-PAGE gel production June 27, 2013: TACC tour - Pickle Research Campus June 28, 2013: Protein characterization - results available next week
June 17, 2013: Agarose gel electrophoresis - PCR I for RE cloning pGFP, Day I transformation of YopH and FtHAP into BL21(DE3) cells, preparation of 500 mL LB for larger scale protein expression June 18, 2013: PCR II for RE cloning pGFP, Day II overnight culture for larger scale protein expression June 19, 2013: Larger scale protein expression YopH in pNIC-Bsa4 June 20, 2013: FF205 training, PCR III for RE cloning pGFP (samples only, no gel) June 21, 2013: Lab closed
Larger scale protein expression YopH in pNIC-Bsa4:
6/18: 2 small 100 mL LB + Kan+ BL21(DE3) with YopH plasmid overnight cultures prepared (≈16 hours in shaking incubator) 6/19: -10 mL of overnight culture added to 500 mL of LB in a 2L flask
Figure 3. Bacterial growth monitored on shaking incubator at 37 °C in ≈30 min. increments
-Sample #0 cell lysate before induction prepared in 1.7 mL centrifuge tube for analytical gel; stored in -20 °C fridge -250 uL IPTG added to 2L flask to induce expression of T7 polymerase -Sample grown for 4 hrs at 37 °C in shaking incubator -Sample #1 cell lysate after induction prepared in 1.7 mL centrifuge tube for analytical gel; stored in -20 °C fridge -JA-10 rotor in centrifuge reserved and pre-cooled; sample from 2L flask transferred to 500 mL cylindrical bottle -Centrifuged sample for 20 min at 6000 g -Disposed supernatant, pellet weight: 1.88 g stored in -80 °C fridge after resuspending pellet in 10 mL buffer
pGFP PCR II:
Figure 2. Agarose gel electrophoresis image pGFP post-PCR II. VDS1 forward/VDS2 reverse primers (lanes 2-5) and M13 forward (-20)/M13 reverse (-27) primers (lanes 6-9). Lane 1 = molecular weight marker 100 bp DNA ladder, lane 2 = sample 1 (0.011 ng/uL pGFP), lane 3 = sample 2 (0.11 ng/uL pGFP), lane 4 = sample 3 (1.1 ng/uL pGFP), lane 5 = sample 4 (0 ng/uL pGFP), lane 6 = sample 5 (0.011 ng/uL pGFP), lane 7 = sample 6 (0.11 ng/uL pGFP), lane 8 = sample 7 (1.1 ng/uL pGFP), and lane 9 = sample 8 (0 ng/uL pGFP). No bands present in lanes 2-9, redo PCR II with different annealing temperature for VDS1/2 primers (53.6 °C instead of 55.6 °C)
pGFP PCR I:
Figure 1. Agarose gel electrophoresis image pGFP post-PCR I. VDS1 forward/VDS2 reverse primers (lanes 2-5) and M13 forward (-20)/M13 reverse (-27) primers (lanes 6-9). Lane 1 = molecular weight marker 100 bp DNA ladder, lane 2 = sample 1 (0.011 ng/uL pGFP), lane 3 = sample 2 (0.11 ng/uL pGFP), lane 4 = sample 3 (1.1 ng/uL pGFP), lane 5 = sample 4 (0 ng/uL pGFP), lane 6 = sample 5 (0.011 ng/uL pGFP), lane 7 = sample 6 (0.11 ng/uL pGFP), lane 8 = sample 7 (1.1 ng/uL pGFP), and lane 9 = sample 8 (0 ng/uL pGFP). No bands present in lanes 2-9, redo PCR with Nanodrop verified pGFP concentration
WEEK 2 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Serena - like the pink! - Good work and good documentation. Do you have your DNA Sequence result to show? - Dr. B June 10, 2013: Primer design overlap assembly June 11, 2013: Agarose gel electrophoresis (check PCR I) June 12, 2013: Midi-Prep, DNA sequencing submission II, ordering target primers June 13, 2013: PCR for RE cloning pGFP/pmCherry June 14, 2013: Optional session
Figure 3. Agarose gel electrophoresis image pGBR22 post-PCR I. Lane 1 = molecular weight marker 1kb DNA ladder, Lane 2 = sample A (1 ng pGBR22), Lane 3 = sample B (5 ng pGBR22), Lane 4 = sample C (25 ng pGBR22), Lane 5 = sample D (zero DNA control), Lanes 6-10 = empty. Lanes 3 & 4 ≈ 1kb.
Midi-Prep Nanodrop:
Figure 2. Trial 2 Midi-Prep Nanodrop spectrophotometer measurement of 2 uL pmCherry elution at λ = 230 nm
Figure 1. Trial 1 Midi-Prep Nanodrop spectrophotometer measurement of 2 uL pmCherry elution at λ = 230 nm
WEEK 1 -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- June 3, 2013: Primer dilution, DNA sequencing submission I June 4, 2013: LB + LB agar plates, Nanodrop plasmid DNA June 5, 2013: Transformation efficiency June 6, 2013: Target discovery (computer lab) June 7, 2013: pGBR22 PCR I
Weeks 9 & 10: October 21 - November 3
October 21-25, 2013: Inhibition Assay I
October 26, 2013: Rice Undergraduate Research Symposium
November 2, 2013: Virtual screening against novel compounds: ChemBridge-diversity3D library
'In house' top-ranking compounds were acquired. Compound 5107893 was tested for inhibition in an enzyme assay. Inhibitory activity was not indicated, as normal nitrocefin hydrolase activity was detected at 486 nm. Additional concentration of compound 5107893 will be tested, along with other top-ranking 'in house' compounds. Top ranking cb306 compounds will be ordered. Virtual screening round against ChemBridgediversity3D library is currently being completed.
Weeks 7 & 8: October 7 - October 20
Have you collected any more data for weeks 7 & 8? Thank you. -Max 10/21/13
October 10, 2013: Synergy HT Biotek Reader to determine optimal [NDM-1]
October 11, 2013: Determining control set of ligands
October 14-18, 2013: LigPrep protocol, GOLD & ICM control ligands docking, virtual screening against novel compounds: cb306 library & 'in house' library
Table 4. Virtual screening against novel compounds: top 7 'in house' library ligands. Best GOLD fitness scores 80-89.55. Compounds ranked 1, 3, 4, and 7 pursued further with favorable LogP values (<2.7).
Table 3. Virtual screening against novel compounds: top 14 cb306 library ligands. Four compounds with GOLD fitness scores >90. All top 14 ligands follow Lipinski's rule of 5. Compounds ranked 1, 2, 5, 9, and 11 pursued further with favorable LogP values (<2.4).
Table 2. ICM validation control docking results. 11 positive control ligands and 5 negative control ligands (ZINC) docked against NDM-1 PDB ID: 4EY2 (chain A). Positive controls are highlighted in green and negative controls are highlighted in pink. Positive and negative control ligands are interspersed, with a few unexpected results. GOLD evaluated control ligands more accurately.
Table 1. GOLD validation control docking results. 11 positive control ligands and 5 negative control ligands (ZINC) docked against NDM-1 PDB ID: 4EY2 (chain A). Positive controls are highlighted in green and negative controls are highlighted in pink. All positive control ligands scored higher than negative control ligands as expected, thus validating GOLD virtual screening software.
Positive control ligands were acquired from PubChem after referring to literature. Negative control ligands were established by the Zinc database after inputting physico-chemical properties of positive control ligands. These ligands were ligprepped and control ligand docking on GOLD & ICM was conducted. NDM-1 PDB ID: 4EY2 was utilized and prepared in GOLD Hermes (2254 Hs added, 227 H2Os deleted). GOLD scored control ligands more accurately than ICM. Virtual screening against novel compounds was conducted using cb306 and 'in house' libraries. Potential inhibitors from both libraries will be acquired and tested for inhibition concurrently.
Weeks 5 & 6: September 23 - October 6
Great Captions and Great analysis. Keep up the nice work. Thank you. -Max 10/07/2013
Good Results Serena, keep up the good work! - Dr. B
September 26-27, 2013: Optimal substrate (nitrocefin) dilution to establish absorbance signal, NDM-1 enzyme assay II
September 30, 2013: NDM-1 enzyme assay III :)
October 1, 2013: NDM-1 1.5 hour enzyme assay IV
October 3-4, 2013: Virtual screening: ICM control docking
Optimal substrate (nitrocefin) dilution was established to register absorbance signal during enzyme assays. 1:5 nitrocefin dilution determined using results from Fig. 1 above (optimal absorbance of 0.5-1.0 at 485 nm). 1:5 substrate dilution was utilized during the second attempt of the enzyme assay. Results in Fig. 2 illustrate that the absorbance change was insignificant during the 10 minute time-based assay, as NDM-1 dilutions created assay conditions with too little enzyme. Enzyme assay III was successful after using 2ug un-diluted NDM-1, which resulted in an expected increase in absorbance through NDM-1 hydrolase activity. NDM-1 enzyme assay results (9/30-10/1/2013) validate reproducibility of assay data for same aliquot of NDM-1. Future research will involve determining optimal [NDM-1] to create a plot with varied substrate concentrations to ultimately determine the Km value. In addition, other aliquots of NDM-1 will be tested to ensure functionality. Lastly, these results indicate that the NDM-1 enzyme purified on 7/31/2013 is indeed functional and can be utilized for enzyme inhibition assays after virtual screening is completed.
Week 3 & 4: September 9 - September 22
Serena - ok good. Show a pretty picture (screenshot is ok) of an enzyme assay graph though. Dr. B 100113
September 12, 2013: Planning NDM-1 enzyme assay
September 13, 2013: Prepared buffers: NDM-1 enzyme assay
September 19, 2013: NDM-1 enzyme assay I with substrate nitrocefin
Preparation of assay buffers
Source: Thomas, P.; Zheng, M.; Wu, S.; Guo, H.; Liu, D.; Xu, D.; Fast, W., Characterization of Purified New Delhi Metallo-β-lactamase-1. Biochemistry 2011, 50, (46), 10102-10113.
Dilute NDM-1 in Buffer A: 50 mM Tris-HCl, pH 7 and 150 mM NaCl
Buffer B: 50 mL 0.1 M Hepes, pH 7 and 20 µM ZnSO4 solution
0.1 mM colorimetric nitrocefin substrate in water (VWR, Radnor, Pennsylvania) from 100 mM stock solution of 5 mg nitrocefin in DMSO
Prepare 0.16-0.33 µg of NDM-1 in total volume of 300 µL and monitor product absorbance at ~485nm
Results of NDM-1 enzyme assay I with nitrocefin:
Spectrophotometric time-based assay (spec: Chipper) resulted in 0 absorbance signal at 486.00 nm for samples:
Buffer B only,
Buffer B + nitrocefin, and
4 µL NDM-1 enzyme + buffer B + nitrocefin.
Full spectrum assay conducted and illustrated 0 absorbance for buffer B only. 0.02 absorbance at ~400 nm and 0 absorbance for all other wavelengths observed for buffer B + nitrocefin sample. In addition, the following samples were tested at full spectrum and had an insignificant absorbance output (maximum 0.01 absorbance at 400 nm):
4 µL NDM-1 + buffer B + nitrocefin,
5 µL NDM-1 + buffer B + nitrocefin,
20 µL NDM-1 + buffer B + nitrocefin,
80 µL NDM-1 + buffer B + nitrocefin,
90 µL NDM-1 + buffer B + nitrocefin, and
Cuvette with Vf = 600 µL of NDM-1 + buffer B + nitrocefin.
These results are worrisome and do not correlate with the cell-based overnight NDM-1 growth assay results. Spectrophotometric signal should result with nitrocefin. Substrate amount may be too minimal to register an absorbance signal. In addition, these results indicate that the NDM-1 enzyme may be inactive (purified 7/31/13). The ExPASy ProtParam tool establishes the estimated half-life of NDM-1: 30 hours (mammalian reticulocytes, in vitro), >20 hours (yeast, in vivo), and >10 hours (Escherichia coli, in vivo). A second enzyme assay should be conducted with increased substrate concentration along with a second spec to elucidate accuracy of Chipper. An additional cell-based overnight NDM-1 growth assay should be completed to test whether the NDM-1 protein is still active. If results are negative, a new batch of purified NDM-1 will need to be utilized.
Serena, ok good. Dr. B 090913
Week 1 & 2: August 26 - September 8
August 29, 2013: Cell-based overnight NDM-1 growth assay
August 30, 2013: Results of overnight NDM-1 growth assay
Results:
2 transformation tubes (+Kan, -Amp): bacterial growth
2 transformation tubes (+ Kan, +Amp): no bacterial growth
2 transformation tubes (+Kan, +Amp, 250 uL NDM-1 protein): medium bacterial growth
2 transformation tubes (+Kan, +Amp, 500 uL NDM-1 protein): large bacterial growth
2 transformation tubes (+Kan, +Amp, 1.5 mL NDM-1 protein): no bacterial growth
Transformation tubes post 16 hours in shaking incubator displayed, for the most part, expected results. However, 1.5 mL NDM-1 protein tubes should have theoretically contained greater bacterial growth than tubes containing 250 uL and 500 uL NDM-1. These results provide confidence that the NDM-1 protein is functional.
WEEK 9 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
July 29, 2013: -----
July 30, 2013: NDM-1 protein expression (day V) - sonication & spin down
July 31, 2013: NDM-1 Ni-NTA affinity purification
August 1, 2013: -----
August 2, 2013: NDM-1 protein characterization; gel dried on 8/5/2013 - K.R.
NDM-1 Protein Nanodrops Post-Purification
Elution 1, Trial 1. Concentration = 0.30 mg/ml, absorbance = 0.299.
Elution 1, Trial 2. Concentration = 0.26 mg/ml, absorbance = 0.264.
Elution 2, Trial 1. Concentration = -0.02 mg/ml, absorbance = -0.019.
Elution 2, Trial 2. Concentration = -0.04 mg/ml, absorbance = -0.038.
WEEK 8 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
July 22, 2013: Results available - cloning #2; starter culture of transformed BL21(DE3) E. coli with pET27b(+) NDM-1(d35): protein expression (day II)
July 23, 2013: NDM-1 protein expression (day III) - K.R.
July 24, 2013: -----
July 25, 2013: Preparation of purification lysis, wash, & elution buffers
July 26, 2013: -----
Table 2. pNIC-Bsa4 cloning round 2 with NDM-1 insert results. Sample #, primer, query coverage (%), identities (#, %), pos/neg clone, and notes provided for each sample submitted to DNA sequencing facility. Information obtained from aligning 2 sequences on NCBI's nucleotide BLAST: core's DNA sequence results (query) and NDM-1 gene DNA sequence (subject). + clone from sample #8!
+ Clone Pairwise Alignments & Chromatograms
pNIC-Bsa4 Cloning #2 with NDM-1 Insert Nanodrops
Colony #1. 2uL pNIC-Bsa4 cloned with NDM-1 insert eluted in 50 uL Elution Buffer. 260/280 = 1.99, 260/230 = 1.70, Concentration = 87.1 ng/uL. Purity ratios are within 0.3 range of 'pure' values. Great concentration - sample submitted to DNA sequencing facility.
Colony #2. 2uL pNIC-Bsa4 cloned with NDM-1 insert eluted in 50 uL Elution Buffer. 260/280 = 1.94, 260/230 = 1.65, Concentration = 107.8 ng/uL. 260/230 ratio could be improved. Excellent concentration - sample submitted to DNA sequencing facility.
Colony #3. 2uL pNIC-Bsa4 cloned with NDM-1 insert eluted in 50 uL Elution Buffer. 260/280 = 2.02, 260/230 = 1.79, Concentration = 79.3 ng/uL. Purity ratios are within 0.2 range of 'pure' values. Great concentration - sample submitted to DNA sequencing facility.
Colony #4. 2uL pNIC-Bsa4 cloned with NDM-1 insert eluted in 50 uL Elution Buffer. 260/280 = 2.04, 260/230 = 1.99, Concentration = 76.0 ng/uL. Purity ratios are within 0.25 range of 'pure' values. Good concentration - sample submitted to DNA sequencing facility.
Colony #5. 2uL pNIC-Bsa4 cloned with NDM-1 insert eluted in 50 uL Elution Buffer. 260/280 = 1.98, 260/230 = 1.72, Concentration = 82.7 ng/uL. Purity ratios are within 0.3 range of 'pure' values. Great concentration - sample submitted to DNA sequencing facility.
Colony #6. 2uL pNIC-Bsa4 cloned with NDM-1 insert eluted in 50 uL Elution Buffer. 260/280 = 2.02, 260/230 = 1.89, Concentration = 69.5 ng/uL. Purity ratios are within 0.2 range of 'pure' values. Good concentration - sample submitted to DNA sequencing facility.
Colony #7. 2uL pNIC-Bsa4 cloned with NDM-1 insert eluted in 50 uL Elution Buffer. 260/280 = 2.00, 260/230 = 1.84, Concentration = 64.4 ng/uL. Purity ratios are within 0.2 range of 'pure' values. Good concentration - sample submitted to DNA sequencing facility.
Colony #8. 2uL pNIC-Bsa4 cloned with NDM-1 insert eluted in 50 uL Elution Buffer. 260/280 = 1.99, 260/230 = 1.53, Concentration = 72.5 ng/uL. 260/230 purity could be improved. Good concentration - sample submitted to DNA sequencing facility.
Colony #9. 2uL pNIC-Bsa4 cloned with NDM-1 insert eluted in 50 uL Elution Buffer. 260/280 = 2.00, 260/230 = 1.92, Concentration = 65.5 ng/uL. Purity ratios are within 0.2 range of 'pure' values. Good concentration - sample submitted to DNA sequencing facility.
NOTE: Samples 1-9 eluted correctly in 50 uL Elution Buffer. All Nanodrops illustrate lower concentration value of 2 trials completed for each colony.
WEEK 7 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
July 14, 2013: Spin down 8 cloning samples
July 15, 2013: Mini-Prep 8 cloning samples
July 16, 2013: Submit 10 cloning samples - DNA sequencing facility
July 17, 2013: Results available - cloning #1
July 18, 2013: Prepared overnight culture of 8 colonies from Masterplate A - cloning #2
July 19, 2013: Submit Midi-Prepped pET27b(+) NDM-1(d35) samples - DNA sequencing facility; spin down, Mini-Prep, submit 9 cloning #2 samples
Table 1. pNIC-Bsa4 cloning round 1 with NDM-1 insert results. Sample #, primer, query coverage (%), identities (#, %), pos/neg clone, and notes provided for each sample submitted to DNA sequencing facility. Information obtained from aligning 2 sequences on NCBI's nucleotide BLAST: core's DNA sequence results (query) and NDM-1 gene DNA sequence (subject). No + clones - perform round 2 of cloning.
NOTE: Changes for round 2 of cloning: elute in accurate amount of Elution Buffer (50 uL); use correct transformation tubes; when eluting - insert buffer directly into the center of column to maximize amount of eluted DNA.
pNIC-Bsa4 Cloning #1 with NDM-1 Insert Nanodrops
Colony #1. 2uL pNIC-Bsa4 cloned with NDM-1 insert eluted in 100 uL Elution Buffer. 260/280 = 1.70, 260/230 = 0.93, Concentration = 70.6 ng/uL. 260/230 purity could be greatly improved. Good concentration - sample submitted to DNA sequencing facility.
Colony #2. 2uL pNIC-Bsa4 cloned with NDM-1 insert eluted in 100 uL Elution Buffer. 260/280 = 1.92, 260/230 = 1.29, Concentration = 41.3 ng/uL. 260/230 purity could be improved. OK concentration - sample submitted to DNA sequencing facility.
Colony #3. 2uL pNIC-Bsa4 cloned with NDM-1 insert eluted in 100 uL Elution Buffer. 260/280 = 2.07, 260/230 = 1.66, Concentration = 35.6 ng/uL. 260/230 and 260/280 ratios could be improved. OK concentration - sample submitted to DNA sequencing facility.
Colony #4. 2uL pNIC-Bsa4 cloned with NDM-1 insert eluted in 100 uL Elution Buffer. 260/280 = 2.04, 260/230 = 1.70, Concentration = 33.2 ng/uL. 260/230 and 260/280 ratios could be improved. OK concentration - sample submitted to DNA sequencing facility.
Colony #5. 2uL pNIC-Bsa4 cloned with NDM-1 insert eluted in 100 uL Elution Buffer. 260/280 = 1.93, 260/230 = 1.52, Concentration = 40.2 ng/uL. 260/230 purity could be improved. OK concentration - sample submitted to DNA sequencing facility.
Colony #6. 2uL pNIC-Bsa4 cloned with NDM-1 insert eluted in 100 uL Elution Buffer. 260/280 = 1.96, 260/230 = 1.81, Concentration = 18.3 ng/uL. 260/230 purity could be improved. Very low concentration - sample submitted to DNA sequencing facility.
Colony #7. 2uL pNIC-Bsa4 cloned with NDM-1 insert eluted in 100 uL Elution Buffer. 260/280 = 1.79, 260/230 = 1.11, Concentration = 45.5 ng/uL. 260/230 purity could be improved. OK concentration - sample submitted to DNA sequencing facility.
Colony #8. 2uL pNIC-Bsa4 cloned with NDM-1 insert eluted in 100 uL Elution Buffer. 260/280 = 1.75, 260/230 = 0.95, Concentration = 56.3 ng/uL. 260/230 purity could be improved. Good concentration - sample submitted to DNA sequencing facility.
NOTE: Samples 1-8 eluted mistakenly in 100 uL Elution Buffer - incorrectly referred to Mini-Prep kit manual. Should elute in 50 uL Elution Buffer. This most likely caused low, non-ideal concentration values in above Nanodrops, particularly for Colony #6.
WEEK 6 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
July 8, 2013: Secondary PCR, 1° and 2° PCR agarose gel electrophoresis verification, pNIC-Bsa4 overnight culture preparation,
Naturally Obsessed: The Making of a Scientist documentary viewing
July 9, 2013: PCR squared reaction, PCR cleanup, pNIC-Bsa4 Midi-Prep - submitted to DNA sequencing facility
July 10, 2013: Cloning: preparation of pNIC-Bsa4 as accepting vector + PCR cleanup, YopH spin down, transformation of DH5α & BL21(DE3) E.coli with pET27b(+) NDM-1(d35) (Day I)
July 11, 2013: pNIC-Bsa4 preparation I gel check, preparation of pNIC-Bsa4 II & III, PCR combination cleanup, gel check II, FPLC - ECDHFR, overnight culture of DH5α E.coli with pET27b(+) NDM-1(d35) (Day II)
July 12, 2013: Spin down & Midi-Prep of DH5α E.coli with pET27b(+) NDM-1(d35) (Day III), cloning: cohesive end generation on PCR insert & accepting vector, annealing & transformation step
July 13, 2013: Master plate & overnight culture of 8 colonies
DNA Sequencing Results: pET27b(+) NDM-1(d35) forward sequence post-Midi-Prep
pET27b(+) NDM-1(d35) Transformed in DH5α E. coli Midi-Prep Nanodrops
Trial 2. 260/280 = 1.94, 260/230 = 3.37, Concentration = 41.5 ng/uL. Purity could be improved. Sample submitted to DNA sequencing facility.
Trial 1. 260/280 = 1.98, 260/230 = 3.15, Concentration = 42.4 ng/uL. Purity could be improved. Sample submitted to DNA sequencing facility.
Preparation #2, 3 of pNIC-Bsa4 as Accepting Vector post-PCR combination cleanup Nanodrops
Trial 2. 260/280 = 1.90, 260/230 = 2.31, Concentration = 65.6 ng/uL. Sample of pNIC-Bsa4 considered 'pure'; includes prep #1 (20 uL), prep #2 (50 uL), prep #3 (50 uL) for a Vf = 120 uL pNIC-Bsa4. Combination sample is usable as an accepting vector.
Trial 1. 260/280 = 1.87, 260/230 = 2.14, Concentration = 63.3 ng/uL. Sample of pNIC-Bsa4 considered 'pure'; includes prep #1 (20 uL), prep #2 (50 uL), prep #3 (50 uL) for a Vf = 120 uL pNIC-Bsa4. Combination sample is usable as an accepting vector.
Note: 260/280 assesses purity of DNA/RNA, optimal value ~1.8 (DNA) and ~2.0 (RNA). 260/230 is a secondary measure of nucleic acid purity, optimal value ~2.0-2.2.
Source: http://www.nanodrop.com/Library/T009-NanoDrop%201000-&-NanoDrop%208000-Nucleic-Acid-Purity-Ratios.pdf
Preparation of pNIC-Bsa4 as Accepting Vector post-PCR cleanup Nanodrops
Trial 2. 260/280 = 1.66, 260/230 = 1.40, Concentration = 18.3 ng/uL. Low concentration & purity requires 2 more rounds of pNIC-Bsa4 preparation.
Trial 1. 260/280 = 1.71, 260/230 = 1.09, Concentration = 19.0 ng/uL. Low concentration & purity requires 2 more rounds of pNIC-Bsa4 preparation.
DNA Sequencing Results: pNIC-Bsa4 Midi-Prep
pNIC-Bsa4 PCR cleanup Nanodrops
Elution 1 Trial 2
Elution 1 Trial 1
PCR^2 cleanup Nanodrops
Elution 1 Trial 2
Elution 1 Trial 1
WEEK 5 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
July 1, 2013: Protocol primer design - pNIC-BSA4 - New Delhi metallo-beta-lactamase-1 (computer lab)
July 2, 2013: Protein characterization - drying the SDS-PAGE gel, Restriction enzyme digest - preparing samples
July 3, 2013: Restriction enzyme digest - agarose gel electrophoresis verification, PCR protocol for pLIC sequencing vectors of pNIC-Bsa4
July 4, 2013: Holiday
July 5, 2013: pLIC PCR - agarose gel electrophoresis verification, Primer overlap PCR with Q5 Polymerase - primer dilutions for assembly step
July 6, 2013: Optional session - Primary PCR
WEEK 4 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
June 24, 2013: Analyze DNA sequence exercise (computer lab)
June 25, 2013: Protein expression scaled up - day 5: sonication & spin down steps
June 26, 2013: Protein purification, SDS-PAGE gel production
June 27, 2013: TACC tour - Pickle Research Campus
June 28, 2013: Protein characterization - results available next week
Elution 1 average absorbance: 0.847
Elution 2 average absorbance: 0.0385
Molar extinction coefficient: 18910 M-1 cm-1
Molecular weight: 33512.8
Beer's law (A=ebc) calculations to determine yield of YopH:
Elution 1 concentration at 280 nm:
Average Abs: (0.838 + 0.856) / 2 = 0.847
Concentration = 0.847/[(18910 M-1 cm-1)(1 cm)]= 4.48 E-5 mol/L (33512.8 g/mol) = 1.50 mg/mL
Yield = (1.50 mg/mL)(5 mL) = 7.51 mg
Elution 2 concentration at 280 nm:
Average Abs: (0.016 + 0.061) / 2 = 0.0385
Concentration = 0.0385/[(18910 M-1 cm-1)(1 cm)]= 2.04 E-6 mol/L (33512.8 g/mol) = 0.0682 mg/mL
Yield = (0.0682 mg/mL)(5 mL) = 0.341 mg
WEEK 3 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
June 17, 2013: Agarose gel electrophoresis - PCR I for RE cloning pGFP, Day I transformation of YopH and FtHAP into BL21(DE3) cells, preparation of 500 mL LB for larger scale protein expression
June 18, 2013: PCR II for RE cloning pGFP, Day II overnight culture for larger scale protein expression
June 19, 2013: Larger scale protein expression YopH in pNIC-Bsa4
June 20, 2013: FF205 training, PCR III for RE cloning pGFP (samples only, no gel)
June 21, 2013: Lab closed
Larger scale protein expression YopH in pNIC-Bsa4:
6/18: 2 small 100 mL LB + Kan+ BL21(DE3) with YopH plasmid overnight cultures prepared (≈16 hours in shaking incubator)
6/19: -10 mL of overnight culture added to 500 mL of LB in a 2L flask
-Sample #0 cell lysate before induction prepared in 1.7 mL centrifuge tube for analytical gel; stored in -20 °C fridge
-250 uL IPTG added to 2L flask to induce expression of T7 polymerase
-Sample grown for 4 hrs at 37 °C in shaking incubator
-Sample #1 cell lysate after induction prepared in 1.7 mL centrifuge tube for analytical gel; stored in -20 °C fridge
-JA-10 rotor in centrifuge reserved and pre-cooled; sample from 2L flask transferred to 500 mL cylindrical bottle
-Centrifuged sample for 20 min at 6000 g
-Disposed supernatant, pellet weight: 1.88 g stored in -80 °C fridge after resuspending pellet in 10 mL buffer
pGFP PCR II:
pGFP PCR I:
WEEK 2 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Serena - like the pink! - Good work and good documentation. Do you have your DNA Sequence result to show? - Dr. B
June 10, 2013: Primer design overlap assembly
June 11, 2013: Agarose gel electrophoresis (check PCR I)
June 12, 2013: Midi-Prep, DNA sequencing submission II, ordering target primers
June 13, 2013: PCR for RE cloning pGFP/pmCherry
June 14, 2013: Optional session
DNA sequencing result II post-MidiPrep:
pGBR22 PCR:
Midi-Prep Nanodrop:
WEEK 1 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
June 3, 2013: Primer dilution, DNA sequencing submission I
June 4, 2013: LB + LB agar plates, Nanodrop plasmid DNA
June 5, 2013: Transformation efficiency
June 6, 2013: Target discovery (computer lab)
June 7, 2013: pGBR22 PCR I
DNA sequencing result I:
Transformation Efficiency = # of colonies / ng of plasmid on each plate
Calculations:
Plate A (1 ng pmCherry plasmid DNA): 1,008 colonies / 1ng = 1,008 colonies/ng
Plate B (5 ng pmCherry plasmid DNA): 3,861 colonies / 5ng = 772.2 colonies/ng
Plate C (25 ng pmCherry plasmid DNA): 1,215 colonies / 25ng = 48.6 colonies/ng